Simple and Divided Leaves in Ferns: Exploring the Genetic Basis for Leaf Morphology Differences in the Genus Elaphoglossum (Dryopteridaceae).

Despite the implications leaves have for life, their origin and development remain debated. Analyses across ferns and seed plants are fundamental to address the conservation or independent origins of megaphyllous leaf developmental mechanisms. Class I KNOX expression studies have been used to understand leaf development and, in ferns, have only been conducted in species with divided leaves. We performed expression analyses of the Class I KNOX and Histone H4 genes throughout the development of leaf primordia in two simple-leaved and one divided-leaved fern taxa. We found Class I KNOX are expressed (1) throughout young and early developing leaves of simple and divided-leaved ferns, (2) later into leaf development of divided-leaved species compared to simple-leaved species, and (3) at the leaf primordium apex and margins. H4 expression is similar in young leaf primordia of simple and divided leaves. Persistent Class I KNOX expression at the margins of divided leaf primordia compared with simple leaf primordia indicates that temporal and spatial patterns of Class I KNOX expression correlate with different fern leaf morphologies. However, our results also indicate that Class I KNOX expression alone is not sufficient to promote divided leaf development in ferns. Class I KNOX patterns of expression in fern leaves support the conservation of an independently recruited developmental mechanism for leaf dissection in megaphylls, the shoot-like nature of fern leaves compared with seed plant leaves, and the critical role marginal meristems play in fern leaf development.

Class I KNOX Is Related to Determinacy during the Leaf Development of the Fern Mickelia scandens (Dryopteridaceae).

Unlike seed plants, ferns leaves are considered to be structures with delayed determinacy, with a leaf apical meristem similar to the shoot apical meristems. To better understand the meristematic organization during leaf development and determinacy control, we analyzed the cell divisions and expression of Class I KNOX genes in Mickelia scandens, a fern that produces larger leaves with more pinnae in its climbing form than in its terrestrial form. We performed anatomical, in situ hybridization, and qRT-PCR experiments with histone H4 (cell division marker) and Class I KNOX genes. We found that Class I KNOX genes are expressed in shoot apical meristems, leaf apical meristems, and pinnae primordia. During early development, cell divisions occur in the most distal regions of the analyzed structures, including pinnae, and are not restricted to apical cells. Fern leaves and pinnae bear apical meristems that may partially act as indeterminate shoots, supporting the hypothesis of homology between shoots and leaves. Class I KNOX expression is correlated with indeterminacy in the apex and leaf of ferns, suggesting a conserved function for these genes in euphyllophytes with compound leaves.

A plastid phylogeny of the fern genus Arachniodes (Dryopteridaceae).

Arachniodes (Dryopteridaceae) is one of the most confusing and controversial fern genera in terms of its circumscription, nomenclature, and taxonomy. Estimates of species number range from 40 to 200. Previous molecular works included only 2-17 accessions representing 2-12 species of Arachniodes and allied genera, leaving most of the Asian species remain unsampled and the infragneric relationships unclear. In this study DNA sequences of seven plastid markers of 343 accessions representing ca. 68 species of Arachniodes (275 accessions), and 64 outgroup accessions from subfam. Dryopteridoideae and subfam. Polybotryoideae were used to infer a phylogeny with maximum likelihood, Bayesian inference, and maximum parsimony approaches. Our major results include: (1) Two species currently assigned in Arachniodes (A. macrostegia and A. ochropteroides are resolved outside of the core Arachniodes making the currently defined Arachniodes polyphyletic, confirming earlier findings; (2) Lithostegia, Leptorumohra, and Phanerophlebiopsis are indeed synonyms of Arachniodes; (3) Leptorumohra is confirmed to be monophyletic, but Phanerophlebiopsis is polyphyletic; (4) The New World species of Arachniodes are confirmed to be not monophyletic with A. denticulata being nested within the Old World species, suggesting that this species is dispersed from the Old World; (5) Arachniodes s.s is resolved into 12 major clades, some of which are further divisable into recognizable subclades and groups, with A. mutica from Japan being resolved as the sister to the rest of the genus; (6) A number of systematic implications of the phylogeny have been suggested; and (7) the genus is estimated to contain ca. 83 species.

Using a multilocus phylogeny to test morphology-based classifications of Polystichum (Dryopteridaceae), one of the largest fern genera.

BACKGROUND: Polystichum (Dryopteridaceae) is probably the third largest fern genus in the world and contains ca. 500 species. Species of Polystichum occur on all continents except Antarctica, but its highest diversity is found in East Asia, especially Southwest China and adjacent regions. Previous studies typically had sparse taxon sampling and used limited DNA sequence data. Consequently, the majority of morphological hypotheses/classifications have never been tested using molecular data. RESULTS: In this study, DNA sequences of five plastid loci of 177 accessions representing ca. 140 species of Polystichum and 13 species of the closely related genera were used to infer a phylogeny using maximum likelihood, Bayesian inference, and maximum parsimony. Our analyses show that (1) Polystichum is monophyletic, this being supported by not only molecular data but also morphological features and distribution information; (2) Polystichum is resolved into two strongly supported monophyletic clades, corresponding to the two subgenera, P. subg. Polystichum and P. subg. Haplopolystichum; (3) Accessions of P. subg. Polystichum are resolved into three major clades: clade K (P. sect. Xiphophyllum), clade L (P. sect. Polystichum), and the HYMASO superclade dominated by accessions of P. sect. Hypopeltis, P. sect. Macropolystichum, and P. sect. Sorolepidium, while those of P. subg. Haplopolystichum are resolved into eight major clades; and (4) The monophyly of the Afra clade (weakly supported), the Australasian clade (weakly supported), and the North American clade (strongly supported) is confirmed. CONCLUSIONS: Of the 23 sections of Polystichum recognized in a recent classification of the genus, four (P. sect. Hypopeltis, P. sect. Neopolystichum, P. sect. Sorolepidium, P. sect. Sphaenopolystichum) are resolved as non-monophyletic, 16 are recovered as monophyletic, and three are monospecific. Of the 16 monophyletic sections, two (P. sect. Adenolepia, P. sect. Cyrtogonellum) are weakly supported and 14 are strongly supported as monophyletic. The relationships of 11 sections (five in P. subg. Haplopolystichum; six in P. subg. Polystichum) are well resolved.

A well-sampled phylogenetic analysis of the polystichoid ferns (Dryopteridaceae) suggests a complex biogeographical history involving both boreotropical migrations and recent transoceanic dispersals.

Intercontinental disjunctions in ferns have often been considered as the result of long-distance dispersal (LDD) events rather than of vicariance. However, in many leptosporangiate groups, both processes appear to have played a major role in shaping current geographical distribution. In this study, we reconstructed the phylogenetic relationships and inferred the ancestral distribution areas of the polystichoid ferns (Cyrtomium, Phanerophlebia, and Polystichum), to evaluate the relative impact of vicariance and LDD on the biogeography of this group. We used a molecular dataset including 3346 characters from five plastid loci. With 190 accessions our taxon coverage was about three times as large as any previous worldwide sampling. Biogeographical analyses were performed using S-DIVA and S-DEC and divergence times were estimated by integrating fossil and secondary calibrations. The polystichoid ferns are a monophyletic clade that may have originated in East Asia during the Eocene, an age much younger than previously estimated. Three transoceanic disjunctions between East Asia and New World were identified in the Paleogene: one for Phanerophlebia during late Eocene (34Ma, 19-51Ma), and two in Polystichum at the Eocene-Oligocene boundary (30Ma, 18-43Ma; 28Ma, 19-39Ma respectively). During the Neogene, further range expansions took place from Asia to Africa, Hawaii, and the Southwestern Indian Ocean region. Our results indicate that early transfers between the Old and the New World are compatible with a boreotropical migration scenario. After evolving in Asia during the Eocene, the polystichoid ferns reached the New World in independent migrations at the Eocene-Oligocene boundary through the boreotropical belt. However, although less likely, the alternative hypothesis of independent transoceanic dispersals from the Old to the New World cannot be ruled out. Further range expansion during the Neogene was most likely the result of long-distance dispersal (LDD).

Significant spatial aggregation and fine-scale genetic structure in the homosporous fern Cyrtomium falcatum (Dryopteridaceae).

Spores of homosporous ferns are small, wind-borne and thus have the potential for long-distance dispersal. This common perception has led to a prediction of near-random spatial genetic structure within fern populations. Spore dispersal and spore bank studies, however, indicate that most spores fall close to the maternal plant (< 5 m), supporting a prediction of significant fine-scale genetic structure (FSGS) within populations. To determine which of these two hypotheses is more likely to occur in nature, we measured inbreeding and quantified the spatial distribution of individuals and allozyme-based genotypes using spatial autocorrelation methods within four populations of the fern Cyrtomium falcatum in southern South Korea. Inbreeding levels were low, and all populations exhibited significant aggregation of individuals and strong FSGS. The present results support the second hypothesis, and the substantial FSGS in C. falcatum could reflect the unique features of most homosporous ferns (outcrossing mating systems that lead a majority of spores to occur at short distances and a very limited dispersal distance of male gametes). Although fern spores are physically analogous to orchid seeds, the intensity of FSGS exhibited in C. falcatum is four times stronger than that in 16 terrestrial orchid species.

Phylogeny, divergence times, and historical biogeography of New World Dryopteris (Dryopteridaceae).

PREMISE OF THE STUDY: Dryopteris is a large, cosmopolitan fern genus ideal for addressing questions about diversification, biogeography, hybridization, and polyploidy, which have historically been understudied in ferns. We constructed a highly resolved, well-supported phylogeny for New World Dryopteris and used it to investigate biogeographic patterns and divergence times. METHODS: We analyzed relationships among 97 species of Dryopteris, including taxa from all major biogeographic regions, with analyses based on 5699 aligned nucleotides from seven plastid loci. Phylogenetic analyses used maximum parsimony, maximum likelihood, and Bayesian inference. We conducted divergence time analyses using BEAST and biogeographic analyses using maximum parsimony, maximum likelihood, Bayesian, and S-DIVA approaches. We explored the monophyly of subgenera and sections in the most recent generic classification and of geographic groups of taxa using Templeton tests. KEY RESULTS: The genus Dryopteris arose ca. 42 million years ago (Ma). Most of the Central and South American species form a well-supported clade which arose 32 Ma, but the remaining New World species are the result of multiple, independent dispersal and vicariance events involving Asia, Europe, and Africa over the last 15 Myr. We identified six long-distance dispersal events and three vicariance events in the immediate ancestry of New World species; reconstructions for another four lineages were ambiguous. CONCLUSIONS: New World Dryopteris are not monophyletic; vicariance has dominated the history of the North American species, while long-distance dispersal prevails in the Central and South American species, a pattern not previously seen in plants.

Evidence of genetic segregation in the apogamous fern species Cyrtomium fortunei (Dryopteridaceae).

In apogamous ferns, all offspring from a parent are expected to be clonal. However, apogamous 'species' frequently demonstrate a large amount of morphological and genetic variations. Cyrtomium fortunei composed of four varieties (C. fortunei var. fortunei, var. clivicola, var. intermedium, and var. atropunctatum), is all reported to be apogamous triploids, but demonstrates large and continuous morphological variation. In previous studies, we showed that considerable genetic diversity was observed in many local populations of the apogamous fern 'species'. We hypothesized that genetic segregation has occurred, because neither sexual type nor intraspecific polyploidy have been observed in C. fortunei in Japan. Of 732 progeny examined (250 gametophytes and 482 sporophytes), obtained from a parental sporophyte whose pgiC genotype was estimated as aab, 11 (4.4%) gametophytes and 8 (1.7%) sporophytes showed a different genotype (aaa) from that of the parent sporophyte. We showed that genetic segregation occurs in apogamous C. fortunei in relatively high frequency. Moreover, we could first show that the segregation frequency in gametophytes is significantly higher than that in sporophytes of the next generation (χ² = 4.90, P = 0.027). It may suggest the existence of deleterious genes, which are expressed during the morphogenesis and growth of sporophytes.

Phylogeny of Chinese Polystichum (Dryopteridaceae) based on chloroplast DNA sequence data (trnL-F and rps4-trnS).

Polystichum is one of the largest and most taxonomically complex fern genera in China. The evolutionary relationships of Chinese Polystichum and related genera, and the relationship between our Polystichum phylogeny and ecogeographic distribution, were tested by the use of DNA sequence data. Fifty-one species of Polystichum and 21 species in allied genera were sequenced for the plastid intergenic spacers rps4-trnS and trnL-F. Maximum parsimony and Bayesian phylogenetic analyses of both individual and combined data sets showed that Chinese Polystichum as commonly recognized was paraphyletic: one clade (the CCPC clade) included Cyrtomidictyum lepidocaulon, two Cyrtogonellum species, three Cyrtomium species, and a small number of Polystichum species usually occurring on limestone. A second clade, Polystichum sensu stricto, included the remainder of the Polystichum species; these often occur on non-limestone substrates. The remaining Cyrtomium species formed the third clade. Three subclades resolved within Polystichum sensu stricto (s.s.) clade do not correspond with recent sectional classifications, and we outline the issues relevant to a new classification for the genus.

Paraphyly of Cyrtomium (Dryopteridaceae): evidence from rbcL and trnL-F sequence data.

Cyrtomium is an Asiatic genus characterized by anastomosing veins with included veinlets, and comprises about 40 species. We sequenced rbcL and trnL-F sequences of 19 species of Cyrtomium and eight species from related genera in order to elucidate a molecular phylogeny of the genus using maximum-parsimony methods. The phylogenetic trees did not agree with traditional classifications. Cyrtomium was resolved as paraphyletic, and a clade including subseries Balansana of Cyrtomium, Cyrtogonellum, Polystichum subacutidens and Cyrtomidictyum (the BCPC clade) and a second one containing Cyrtomium sensu stricto were monophyletic. The results also implied that: (1) C. uniseriale was synonymous with C. balansae; (2) C. falcatum was likely the female parent of C. devexiscapulae; and (3) based on the rbcL and trnL-F sequence data, C. nephrolepioides and C. grossum were the female parents of C. shingianum and C. chingianum, respectively, although other evidence is needed for the confirmation of this hypothesis.

Molecular phylogenetics and historical biogeography of Hawaiian Dryopteris (Dryopteridaceae).

The fern genus Dryopteris (Dryopteridaceae) is represented in the Hawaiian Islands by 18 endemic taxa and one non-endemic, native species. The goals of this study were to determine whether Dryopteris in Hawai'i is monophyletic and to infer the biogeographical origins of Hawaiian Dryopteris by determining the geographical distributions of their closest living relatives. We sequenced two chloroplast DNA fragments, rbcL and the trnL-F intergenic spacer (IGS), for 18 Hawaiian taxa, 45 non-Hawaiian taxa, and two outgroup species. For individual fragments, we estimated phylogenetic relationships using Bayesian inference and maximum parsimony. We performed a combined analysis of both cpDNA fragments employing Bayesian inference, maximum parsimony, and maximum likelihood. These analyses indicate that Hawaiian Dryopteris is not monophyletic, and that there were at least five separate colonizations of the Hawaiian Islands by different species of dryopteroid ferns, with most of the five groups having closest relatives in SE Asia. The results suggest that one colonizing ancestor, perhaps from SE Asia, gave rise to eight endemic taxa (the glabra group). Another colonizing ancestor, also possibly from SE Asia, gave rise to a group of five endemic taxa (the exindusiate group). Dryopteris fusco-atra and its two varieties, which are endemic to Hawai'i, most likely diversified from a SE Asian ancestor. The Hawaiian endemic Nothoperanema rubiginosum has its closest relatives in SE Asia, and while the remaining two species, D. wallichiana and D. subbipinnata, are sister species, their biogeographical origins could not be determined from these analyses due to the widespread distributions of D. wallichiana and its closest non-Hawaiian relative.

Apomixis in the interspecific triploid hybrid fern Cornopteris christenseniana (Woodsiaceae).

Cornopteris christenseniana is a "sterile" interspecific triploid hybrid of diploid C. crenulatoserrulata and tetraploid C. decurrenti-alata. Morphological and cytological studies show that, of 41 young plants of Cornopteris that have been propagated naturally in the Fern Garden of the Botanical Gardens, University of Tokyo, 30 plants are the sterile C. christenseniana, 10 are fertile C. decurrenti-alata and 1, fertile C. crenulatoserrulata. This proportion supports the view that the young plants of C. christenseniana are derived from spores of reproductively mature plants of the species cultivated. Cytogenetic observations and culture experiments show that C. christenseniana produces normal spores in various proportions in some sporogenetic pathways that are aberrant from the ordinary process in sexual and apomictic ferns. Under culture conditions, normal spores germinate in rough proportion to the frequency of normal spores, and sporophytes are apogamously produced in rough proportion to the frequency of spore germination. As a whole, the rates of spore germination and apogamous sporophyte development vary according to the specific plant. Taken together, these observations suggest that C. christenseniana is an incipient apomict.

Electrophoretic evidence for homoeologous chromosome pairing in the apogamous fern species Dryopteris nipponensis (Dryopteridaceae).

Segregation of genotypes through homoeologous chromosome pairing in the apogamous species Dryopteris nipponensis was tested by electrophoretic analysis. Of 284 progeny examined (250 gametophytes and 34 sporophytes), from the parental sporophyte with the Pgi-2 genotype abc, five showed different genotypes from that of the parent (three aac, one bbc and one bcc). This is the first evidence for genetic segregation in the progeny of apogamous fern species.